Search Results (67)

Dengue virus (DENV), an important mosquito-borne orthoflavivirus, represents a growing global threat due to its geographic expansion and recent outbreaks worldwide. In resource-limited endemic settings, the development of affordable diagnostic assays is needed. In this study, we developed and validated a colorimetric reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the detection of DENV type 3 (DENV-3) using 95 previously diagnosed clinical samples from Southeastern Mexico. Primers targeting the 3′ untranslated region (3′ UTR) of DENV-3 were designed, and assay conditions were standardized. The colorimetric RT-LAMP DENV-3 system achieved a preliminary limit of detection of 1 × 10³ copies per reaction, with 90.7% sensitivity and 100% specificity. The colorimetric format enabled visual readout without specialized equipment, supporting its potential applicability in point-of-care and resource-limited settings. The developed colorimetric RT-LAMP detection for DENV-3 is intended as a rapid screening/triage tool that can trigger confirmatory testing or public-health actions. Full article

Background/Objectives: Chimpanzee adenoviral-vectored vaccines have proven to be both safe and effective, with a manufacturing and distribution pipeline capable of rapid global supply, as demonstrated during the COVID-19 pandemic. Yellow fever is a mosquito-borne viral hemorrhagic disease endemic in parts of Africa and Latin America, and although an effective live attenuated vaccine exists, its use is limited by safety and eligibility restrictions. Moreover, large outbreaks continue to expose critical challenges, such as an insufficient vaccine supply, reliance on fractional dosing, and slow and difficult-to-scale manufacturing processes. Here, we report the design, development and in vivo immunogenicity of multiple yellow fever virus (YFV) antigen constructs based on the pre-membrane (prM) and envelope (E) proteins—with or without the transmembrane domain (TM or ΔTM)—delivered using the ChAdOx1 adenoviral vector. Methods: Four ChAdOx1 YF vaccines were developed, and immunogenicity was evaluated. The efficacy of the full-length YF envelope vaccine was also tested in Balb/c mice. Results/Conclusions: In contrast to previously described orthoflavivirus vaccines on the same platform, the full-length antigen elicited superior immunogenicity and conferred protection against intracranial challenge with the YF17D virus in mice. Notably, this protection was comparable to that induced by the licensed YF17D vaccine, highlighting the promise of this platform as a next-generation yellow fever vaccine candidate. Full article

The zoonotic Orthoflavivirus Usutu virus (USUV) is distributed throughout Germany, putting hosts at a considerable risk of infection nationwide. Besides birds as reservoir hosts, a broad range of accidental hosts is suspected. However, there are few reports documenting the progression of USUV-associated disease. This case report describes the course of fatal USUV infections in three harbor seals (Phoca vitulina) from a rescue center on the North Sea coast in Germany. Corresponding samples were analyzed using (histo-)pathological, immunohistochemical, molecular and phylogenetic methods. The most prevalent findings in clinically affected animals were neurological signs and non-suppurative encephalitis. All animals were found dead or had been euthanized due to animal welfare reasons within 30 h after the onset of clinical signs. Blood samples taken from another 37 young harbor seals from the same rescue center in the same year revealed two further asymptomatic USUV RNA and antibody-positive animals. The sequences were found to belong to USUV lineages Europe 2 and Africa 3, which are known to circulate in birds in Germany. This case report highlights the importance of USUV as a potential diagnosis for neurological impairments in marine mammals and documents the first cases of USUV infection in harbor seals. Full article

Dengue virus is an important human pathogen that infects over 400 million people each year. Despite its global health significance, several essential aspects of the viral replication mechanism remain poorly understood. Flaviviruses carry out asymmetric viral RNA synthesis, wherein positive-strand RNA is synthesized in excess over negative-strand RNA. The template for positive-strand synthesis is the negative strand in a double-stranded RNA intermediate, yet little is known about how positive-strand RNA synthesis is initiated. Orthoflaviviruses, including dengue virus, require an RNA promoter, stem–loop A (SLA) at the 5′ end of the viral genome for negative-strand RNA synthesis. Consequently, a complementary stem–loop structure is predicted at the 3′ end of the negative strand (3′SLA), where positive-strand synthesis is initiated. To understand the functional role of 3′SLA, we investigated its structure and examined its interaction with the viral replication proteins NS5 polymerase and NS3 helicase. NS5 and NS3 differentially recognize the stem–loop structures of the positive and negative strands (5′SLA and 3′SLA, respectively), yet NS5 polymerase efficiently synthesizes RNA from both 5′SLA- and 3′SLA-containing templates. We further show that the stable 5′ and 3′SLA elements readily form a duplex that mimics the replication intermediate under our in vitro conditions. Both NS5 and NS3 showed reduced binding to this dsRNA intermediate and NS3 was unable to unwind it, suggesting that additional factors may be required to regulate viral replication in infected cells. Full article

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic orthoflavivirus that poses a significant global health threat. It causes severe neuroinflammatory disease in humans and reproductive failure in swine. Because of the broad host range and cell tropism of JEV, identifying animal cell lines resistant to infection has been a persistent challenge. In this study, we demonstrate that Madin–Darby bovine kidney (MDBK) cells are resistant to JEV infection yet remain fully permissive to viral replication when transfected with viral genomic RNA. Using immunoblotting, immunofluorescence, and flow cytometry, we show that MDBK cells, unlike the highly susceptible baby hamster kidney (BHK-21) cells used as controls, do not support viral entry but sustain all post-entry stages of the replication cycle. Further investigation confirmed that MDBK cells possess a functional clathrin-mediated endocytic pathway, as evidenced by their susceptibility to bovine viral diarrhea virus, which relies on clathrin-dependent endocytosis for host cell entry. These findings establish MDBK cells as a nonsusceptible cell line for JEV entry despite intact endocytic function, providing a valuable platform for studying virus–host cell interactions and for identifying and validating host cell entry factors, a major challenge in JEV research. Full article

Advances in diagnostic techniques, along with environmental changes driven by human activity, have intensified the surveillance and monitoring of virus and arbovirus circulation on the Amazon. These efforts have increased the detection of insect-specific viruses in field-collected hematophagous arthropods. This study reports the first isolation of the Aguas Brancas virus from mosquitoes collected in the Brazilian Amazon and in a rural area of Brasília, Federal District, Brazil. Arthropods of the family Culicidae, genus Limatus durhamii, were collected at ground level in forest fragments. Sample BEAR812610 originated from Ananindeua, Pará, within the Evandro Chagas Institute’s grounds, and sample BEAR839941 from a forest fragment in Brasília (Ceilândia—Núcleo Rural Boa Esperança, Site B4). Specimens were identified to the species/genus level, macerated, and the supernatant inoculated into C6/36 and Vero cell cultures for viral isolation. The presence of arboviruses was determined by indirect immunofluorescence using antibodies against major arbovirus groups. Positive samples were sequenced for nucleotide and amino acid identification, and phylogenetic analysis confirmed the virus as belonging to the genus Orthoflavivirus. This represents the first report of the isolation and characterization of the insect-specific Aguas Brancas virus. Full article

The Orthoflavivirus genus includes a variety of human-pathogenic, mosquito-borne flaviviruses (MBFs) including dengue, Zika, and West Nile viruses, which pose significant global public health threats. Insect-specific flaviviruses (ISFs) are another group within the genus that exclusively replicate in mosquitoes and are incapable of infecting vertebrates. ISFs have recently attracted growing research interest due to their potential applications in vaccine development. In addition, multiple studies have demonstrated that prior infection with ISFs such as Palm Creek virus and Binjari virus can suppress subsequent infection with human-pathogenic MBFs. This phenomenon, known as superinfection exclusion (SIE), opens the avenue for the potential applications of ISFs in MBF transmission control. This prompted a growing number of studies into ISFs and their interactions with MBFs in mosquito hosts. In this review, we provide an overview on ISFs, with a particular emphasis on the capacity of different ISFs to cause SIE, the current insights into the mechanisms of this phenomenon, and the potential use of ISFs in the SIE-based biocontrol strategies. Full article

Arthropod-borne orthoflaviviruses, including dengue, Zika, Japanese encephalitis, yellow fever and West Nile viruses, pose a significant global public health threat, causing hundreds of millions of infections annually with severe clinical symptoms. However, the lack of effective vaccines and antiviral drugs, coupled with the biosafety risks associated with handling live highly pathogenic strains, hinders progress in antiviral research. Pseudovirus technology, which uses single-round infectious viral particles lacking replication competence, has thus gained prominence as a safe and versatile tool for antiviral research. This review systematically summarizes the construction, optimization, and applications of orthoflavivirus pseudoviruses in antiviral research. The primary construction strategies of orthoflavivirus pseudoviruses rely on multi-plasmid co-transfection of viral replicons and structural protein expression vectors, leveraging the host cell secretory pathway to mimic natural viral assembly and maturation. The core applications of pseudovirus technology are highlighted, including high-throughput screening and detection of neutralizing antibodies, identification of antiviral drugs targeting viral entry or replication, and evaluation of vaccine immunogenicity. Despite these strengths, the approach still faces limitations, such as incomplete simulation of native viral structures and batch-to-batch titer variability, which may affect the physiological relevance of findings. In summary, orthoflavivirus pseudovirus technology has become an essential platform in both basic virology research and translational medicine, providing critical insights and tools in the ongoing fight against arthropod-borne orthoflaviviruses diseases. Full article

Interest in Zika virus (ZIKV, Orthoflavivirus zikaense) evolution and pathogenicity has attracted the attention of a wider circle of the research community ever since ZIKV emerged on the South American continent in 2015, after more or less island hopping across the Pacific Ocean from Southeast Asia on its way from Africa where it was first described in Uganda in 1947 [...] Full article

Vector-borne diseases account for over 17% of reported infectious diseases worldwide and are associated with approximately 700,000 deaths annually. The main vectors include mosquitoes, moths, sand flies, black flies, and ticks. Ticks deserve special attention because they transmit a wide range of pathogens, including viruses of major medical importance, such as tick-borne encephalitis virus (Orthoflavivirus encephalitidis) and Crimean-Congo hemorrhagic fever virus (Orthonairovirus haemorrhagiae), as well as animal-borne pathogens, such as African swine fever virus (Asfivirus haemorrhagiae). Recent advances in next-generation sequencing have expanded the ability to detect and characterize tick-borne viruses, revealing increasing viral diversity. However, for many of these viruses, aspects such as pathogenic potential, main vectors, and natural hosts remain unclear. To address this gap, we conducted an integrative literature review using the PubMed, SciELO, BVSalud, and Patuá-IEC databases. We analyzed 336 articles addressing various species of tick-borne viruses. The Flaviviridae, Phenuiviridae, and Nairoviridae families were the most frequently identified among the viral agents detected. Furthermore, we identified that as-yet-unclassified viruses have been frequently detected in different tick species, which sparks significant interest in investigating their potential interactions and public health implications. Investigating viral agents in tick populations is crucial for understanding viral diversity and assessing potential public health risks, especially in the current context of climate change. Full article

Background: Latvia is one of the most endemic tick-borne encephalitis (TBE) countries in Europe. However, assessing TBE seroprevalence and true infection rates has been challenging. Current diagnostics cannot reliably distinguish between immune responses induced by natural infection from those induced by vaccination, especially in TBE endemic countries with recommended immunisation programmes. A recently developed ELISA targeting antibodies against TBEV non-structural protein 1 (NS1) offers improved specificity for natural infection and can differentiate among three clinically relevant TBEV subtypes. Methods: We conducted a cross-sectional TBEV seroprevalence study in the Latvian population during 2019–2022, consisting of two pools: 1020 residents living in different regions of Latvia and 200 random Latvian Biobank blood samples. We used the standard anti-TBEV IgG ELISA (VIDITEST, VIDIA, Czech Republic) for the screening and detection of TBEV (whole virus) IgG antibodies and the newly established research-use anti-TBEV NS1 IgG ELISA for the detection of subtype-specific TBEV NS1 IgG antibodies against three virus subtypes: European, Siberian and Far Eastern. Results: The total TBEV seroprevalence among 1020 residents was 39.7%, representing all age cohorts of the population from all regions of Latvia. In total, 33.4% of the enrolled population were vaccinated against TBE with at least one dose of the TBE vaccine. Among the unvaccinated population, 16.3% had positive TBEV-specific IgG antibodies by standard ELISA. On the contrary, NS1-specific antibodies, indicating past natural infection, were detected in only 4.3% of the overall study population. Subtype differentiation revealed infections from all three major TBEV subtypes present in Latvia. Conclusions: In conclusion, this population-based study highlights a high risk of TBE in Latvia, with substantial seroprevalence even among unvaccinated individuals. The NS1-based ELISA enhances the accuracy of TBE surveillance and offers important clinical utility by facilitating more reliable diagnosis and case classification, regardless of vaccination status. Full article

The genus Orthoflavivirus (family Flaviviridae) comprises several important pathogens that are widespread across the globe, often co-circulating in many regions. In Austria, the closely related mosquito-borne West Nile (WN) and Usutu (USU) viruses have been detected since the early 2000s. Orthoflavivirus-neutralizing antibodies primarily target the major envelope protein E. However, due to their antigenic relationship, recurring contacts with different orthoflaviviruses can lead to the induction of broadly cross-reactive E-specific antibodies. These can pose a problem in the diagnosis and differentiation of orthoflavivirus infections. Therefore, we established immunological assays based on the non-structural protein 1 (NS1) to differentiate infections caused by WN and USU viruses. The NS1 protein is secreted during acute infection, and NS1-specific antibodies have been reported to be less cross-reactive than those against E. Using sera from individuals with a confirmed WN or USU virus infection, it was possible to distinguish between the two virus infections with high accuracy, specifically when IgM and IgG results were combined. Full article

Cacipacoré virus (CPCV) is an understudied orthoflavivirus with significant gaps in research that hinders our understanding of its ecology, host range, and potential public health impact. A notable barrier to filling this gap is the absence of standardized methodologies for viral quantification, such as plaque-forming and focus-forming assays. This short communication outlines the development of optimized protocols for CPCV titration in two cell lines, Vero CCL-81 and BHK CCL-10, using several overlay and time point conditions. These methodologies address the need for robust quantitative tools to advance research on CPCV and its implications for human and animal health, fostering progress in the surveillance and understanding of this underexplored arbovirus. Full article

The cellular membrane is a dynamic structure composed of lipids and proteins organized into specialized domains that facilitate interactions between extracellular molecules and the intracellular environment. Tetraspanins are a family of transmembrane proteins involved in diverse cellular processes, including membrane stabilization and fusion, endocytosis, extracellular vesicle formation, and the organization of proteins and lipids at specific membrane sites known as Tetraspanin-Enriched Microdomains (TEMs). These lipid–protein interactions play a critical role in the replicative cycle of Orthoflavivirus, including dengue, Zika, and West Nile, by facilitating viral entry, replication, assembly, and egress. In addition, tetraspanins also regulate the biogenesis and function of extracellular vesicles, contributing to viral dissemination, persistent infection, and immune evasion. This review summarizes the current knowledge on the structural and functional aspects of tetraspanins, their interplay with lipids, and their emerging roles in the Orthoflavivirus replicative cycle. We also discuss how these insights may inform the development of antiviral strategies targeting membrane organization and virus–host interactions. Full article

The Orthoflavivirus genus belongs to the Flaviviridae family. Orthoflaviviruses include major clinically relevant arthropod-borne human viruses such as Dengue, Zika, yellow fever, West Nile and tick-borne encephalitis virus. These viruses pose an increasing threat to global health due to the expansion of arthropod habitats, urbanization, and climate change. While vaccines have been developed for certain orthoflaviviruses with varying levels of success, critical challenges remain in achieving broadly deployable vaccines that combine a robust safety profile with durable immunity against many current and emerging orthoflaviviruses. This review provides a snapshot of established and emerging vaccine platforms against orthoflaviviruses, with a particular emphasis on those leveraging the envelope glycoprotein E as the primary antigen. We examine the strengths and disadvantages of these different platforms in eliciting safe, durable, and robust orthoflavivirus immunity, and discuss how specific attributes such as multivalency, authentic epitope presentations, and logistical practicality can enhance their value in preventing orthoflavivirus infection and disease. Full article